Comparison of three PCR-based methods to detect a Piedmontese cattle point mutation in the Myostatin gene

Abstract

Despite many recent advances in single nucleotide polymorphism (SNP) genotyping technologies, there is still the need for methodologies with a reasonable throughput. In this study, we compared three PCR-based methods for SNP detection: (1) a conventional PCR-based allele detection system with fluorescent genotyping technology, (2) a SNaPshot methodology by single nucleotide primer extension and, (3) a real-time PCR-based method by allele-specific minor groove-binder probes. These three methodologies were used to analyze 104 meat samples of a particular Italian cattle breed known for producing excellent quality meat and for a characteristic increased development of muscle mass, caused by a point mutation (C313Y) in the Myostatin gene. The analysis revealed 98 samples to be homozygous (mh/mh) and five to be heterozygous (mh/+) for the mutation whereas one sample resulted to be homozygous for the wild type (+/+). The results obtained with the three different assays were consistent. Overall, all three methodologies proved to be efficient for allelic discrimination studies; however, real-time PCR was faster and allowed to genotype up to 96 samples in a single step, minimizing the number of steps required for samples manipulation.

Keywords

• cattle
• Myostatin gene
• SNP detection
• genotyping